Binding of 1alpha,25-dihydroxyvitamin D(3) to annexin II: effect of vitamin D metabolites and calcium

We have recently reported that annexin II serves as a membrane receptor for 1alpha,25-(OH)(2)D(3) and mediates the rapid effect of the hormone on intracellular calcium. The purpose of these studies was to characterize the binding of the hormone to annexin II, determine the specificity of binding, and assess the effect of calcium on binding. The binding of [(14)C]-1alpha,25-(OH)(2)D(3) bromoacetate to purified annexin II was inhibited by 1alpha, 25-(OH)(2)D(3) in a concentration-dependent manner. Binding of the radiolabeled ligand to annexin II was markedly diminished by 1alpha, 25-(OH)(2)D(3) at 24 microM, 18 microM, and 12 microM and blunted by 6 microM and 3 microM. At a concentration of 12 microM, 1beta, 25-(OH)(2)D(3) also diminished the binding of [(14)C]-1alpha, 25-(OH)(2)D(3) bromoacetate to annexin II, but cholecalciferol, 25-(OH)D(3), and 24,25-(OH)(2)D(3) did not. Saturation analyses of the binding of [(3)H]-1alpha,25-(OH)(2)D(3) to purified annexin II showed a K(D) of 5.5 x 10(-9) M, whereas [(3)H]-1beta,25-(OH)(2)D(3) exhibited a K(D) of 6.0 x 10(-9) M. Calcium, which binds to the carboxy terminal domain of annexin II, had a concentration-dependent effect on [(14)C]-1alpha,25-(OH)(2)D(3) bromoacetate binding to annexin II, with 600 nM calcium being able to inhibit binding of the radiolabeled analog. The inhibitory effect of calcium was prevented by EDTA. Homocysteine, which binds to the amino terminal domain of annexin II, had no effect on the binding of the bromoacetate analog to the protein. The data indicate that 1alpha,25-(OH)(2)D(3) binding to annexin II is specific and suggest that the binding site may be located on the carboxy terminal domain of the protein. The ability of 1beta,25-(OH)(2)D(3) to inhibit the binding of [(14)C]-1alpha, 25(OH)(2)D(3) bromoacetate to annexin II provides a biochemical explanation for the ability of the 1beta-epimer to inhibit the rapid actions of the hormone in vitro.     

Metabolism of 1alpha,25-dihydroxyvitamin D(3) in vitamin D receptor-ablated mice in vivo

The metabolism of 1alpha,25-dihydroxyvitamin D(3) was studied in vitamin D receptor-ablated mice following the administration of a physiological dose of 1alpha,25-dihydroxy-[26,27-(3)H]vitamin D(3). The degradation of 1alpha,25-dihydroxy-[26,27-(3)H]vitamin D(3) in the vitamin D receptor null mutant mice was substantially reduced compared to the wild-type control mice. At 24 h postadministration of radiolabeled 1alpha,25-dihydroxyvitamin D(3) more than 50% of the radioactivity was recovered unmetabolized, whereas in wild-type mice nearly all of the 1alpha,25-dihydroxy-[26,27-(3)H]vitamin D(3) was degraded. In wild-type mice three polar metabolites other than 1alpha,25-dihydroxyvitamin D(3) were detected and identified on straight-phase and reverse-phase high-performance liquid chromatography as 1alpha,24(R),25-trihydroxy-[26,27-(3)H]vitamin D(3), 1alpha,25(S),26-trihydroxy-[26,27-(3)H]vitamin D(3), and (23S, 25R)-1alpha,25-dihydroxy-[(3)H]vitamin D(3)-26,23-lactone. Only one metabolite was detected in the plasma and kidneys of vitamin D receptor null mutant mice at 3 h following an intrajugular dose of 1alpha,25-dihydroxy-[26,27-(3)H]vitamin D(3). This metabolite was clearly identified as 1alpha,25(S),26-trihydroxy-[26,27-(3)H]vitamin D(3) by comigration on two HPLC systems and periodate cleavage reaction. At 6, 12, and 24 h postinjection 1alpha,24(R), 25-trihydroxy-[26,27-(3)H]vitamin D(3) was also detected at low levels in plasma, kidneys, and liver of some but not all mutant mice. The presence of 25-hydroxyvitamin D(3)-24-hydroxylase mRNA in the kidneys of these vitamin D receptor null mutant mice was confirmed by ribonuclease protection assay.     

The effects of 1alpha,24(S)-dihydroxyvitamin D(2) analog on cancer cell proliferation and cytokine expression

It is well established that 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)), the active metabolite of vitamin D, plays a role in regulating proliferation and differentiation of cells, in addition to its classic function in mineral homeostasis. Recent studies have also provided evidence for the involvement of 1alpha,25(OH)(2)D(3) in regulating the immune system. However, therapeutic application of 1alpha,25(OH)(2)D(3) to hyperproliferative diseases such as cancer, or for immunologic purposes, is thwarted by its hypercalcemic activity. In order to overcome this obstacle, analogs of 1alpha,25(OH)(2)D(3) have been produced that exhibit decreased hypercalcemic activity while retaining the growth and immunologic regulating properties. In the present study, the efficacy of 1alpha,24(S)-dihydroxyvitamin D(2) (1alpha,24(S)(OH)(2)D(2)), a vitamin D(2) analog, in restraining cell proliferation was compared to that of 1alpha,25(OH)(2)D(3). In parallel studies, cancer cell lines were grown in increased concentrations (10(-10)-10(-7) M) of each compound for various incubation periods (1-4 days). Growth was assessed by measuring [(3)H]thymidine incorporation. The results revealed that 1alpha,24(S)(OH)(2)D(2) significantly inhibits proliferation to an extent similar to that observed for 1alpha,25(OH)(2)D(3). Moreover, incubating the human leukemia cell line, HL-60, with 1alpha,24(S)(OH)(2)D(2) resulted in an induction of differentiation of these promyelomonocyte cells into monocyte-macrophage-like cells, in a manner similar to that observed with 1alpha,25(OH)(2)D(3). Using a Western procedure, it was also shown that 1alpha,24(S)(OH)(2)D(2) like 1alpha,25(OH)(2)D(3) enhances the expression of vitamin D receptors (VDR) in the rat osteosarcoma cell line, ROS 17/2.8. The expression of tumor necrosis factor (TNF) alpha (TNF-alpha) in human peritoneal macrophages (HPM) obtained from uremic patients treated with continuous ambulatory peritoneal dialysis (CAPD) was found to be regulated by 1alpha,25(OH)(2)D(3) as well as by 1alpha,24(S)(OH)(2)D(2). Incubations of HPM with 1alpha,25(OH)(2)D(3) or 1alpha,24(S)(OH)(2)D(2), have inhibited the expression of TNF-alpha on both mRNA and protein levels. These results suggest that 1alpha,25(OH)(2)D(3) has a role in controlling the rate of inflammation in the peritoneal cavity of CAPD treated patients. Since 1alpha,24(S)(OH)(2)D(2) does not cause hypercalcemia, the present results encourage the possible use of this vitamin D(2) analog in the treatment of cancer and hyper-inflammatory diseases.     

Mechanistic studies to evaluate the enhanced antiproliferation of human keratinocytes by 1alpha,25-dihydroxyvitamin D(3)-3-bromoacetate, a covalent modifier of vitamin D receptor, compared to 1alpha,25-dihydroxyvitamin D(3).

1alpha,25-Dihydroxyvitamin D(3)-3-bromoacetate (1, 25(OH)(2)D(3)-3-BE), an affinity labeling analog of 1alpha, 25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), displayed stronger antiproliferative activities than 1,25(OH)(2)D(3) at 10(-10)-10(-6) M dose levels in cultured human keratinocytes (CHK). Additionally, preincubation of the cells with 10(-6) M 1,25(OH)(2)D(3), followed by treatment with various doses of 1,25(OH)(2)D(3)-3-BE, resulted in a significantly stronger antiproliferative activity by the mixture than individual reagents at every dose level. To search for a mechanism of this observation, we determined that [(14)C]1, 25(OH)(2)D(3)-3-BE covalently labeled human recombinant 1alpha, 25-dihydroxyvitamin D(3) receptor (reVDR) swiftly (<1 min) with a 1:1 stoichiometry and induced conformational changes (in VDR) that are different from 1,25(OH)(2)D(3), by limited tryptic digestion. Furthermore, a protein band, corresponding to reVDR, was specifically labeled by [(14)C]1,25(OH)(2)D(3)-3-BE in CHK extract, indicating that VDR is the main target of [(14)C]1, 25(OH)(2)D(3)-3-BE. The above-mentioned observations suggest that a rapid covalent labeling of VDR in CHK might alter the interaction between the holo-VDR and 1,25(OH)(2)D(3)-controlled genes. Furthermore, we observed that 1,25(OH)(2)D(3)-3-BE significantly decreased the binding of VDR to human osteocalcin vitamin D responsive element (hOCVDRE), as well as the dissociation rate of VDR from hOCVDRE, compared with 1,25(OH)(2)D(3) in COS-1 cells, transiently transfected with a VDR construct. Additionally, 1, 25(OH)(2)D(3)-3-BE was found to be more potent in inducing 1alpha, 25-dihydroxyvitamin D(3)-24-hydroxylase (24-OHase) promoter activity and mRNA expression in keratinocytes. The accumulation of 24-OHase message was also prolonged by the analog. Collectively these results indicated that rapid covalent labeling of VDR in keratinocytes (by 1, 25(OH)(2)D(3)-3-BE) might result in the conversion of apo-VDR to a holo-form, with a conformation that is different from that of the 1, 25(OH)(2)D(3)-VDR complex. This resulted in an enhanced stability of the 1,25(OH)(2)D(3)-3-BE/VDR-VDRE complex and contributed to the amplified antiproliferative effect of 1,25(OH)(2)D(3)-3-BE in keratinocytes.     

Stimulatory effect of 1 alpha, 25-dihydroxyvitamin D3 on the formation of skin tumors in mice treated chronically with 7,12-dimethylbenz[a]anthracene

The effect of 1 alpha, 25-dihydroxyvitamin D3 (1 alpha, 25-(OH)2D3) and its 24,24-difluoro analog on the formation of skin tumors in mice was evaluated in a complete carcinogenesis model using 7,12-dimethylbenz[a]anthracene (DMBA) as the carcinogen. Twice weekly topical application of 0.25-0.50 nmol of 1 alpha, 25-(OH)2D3 or 0.05-0.10 nmol of the difluoro analog of 1 alpha, 25-(OH)2D3 1 hour prior to treatment with 50 nmol DMBA stimulated tumor formation several fold compared to animals receiving DMBA alone. Topical application of 0.50 nmol of 1 alpha, 25-(OH)2D3 24 hours after treatment with DMBA, or half of this dose of the vitamin D3 metabolite, applied 1 hour before and 24 hours after treatment with DMBA, also stimulated tumor formation several fold. These results are in marked contrast to the potent inhibitory effect of 1 alpha, 25-(OH)2D3 and its difluoro analog on the formation of skin tumors in mice promoted by 12-O-tetradecanoylphorbol-13-acetate.     

Binding of 1α,25‐dihydroxyvitamin D3 to annexin II: Effect of vitamin D metabolites and calcium

We have recently reported that annexin II serves as a membrane receptor for 1α,25‐(OH)₂D₃ and mediates the rapid effect of the hormone on intracellular calcium. The purpose of these studies was to characterize the binding of the hormone to annexin II, determine the specificity of binding, and assess the effect of calcium on binding. The binding of [¹⁴C]‐1α,25‐(OH)₂D₃ bromoacetate to purified annexin II was inhibited by 1α,25‐(OH)₂D₃ in a concentration‐dependent manner. Binding of the radiolabeled ligand to annexin II was markedly diminished by 1α,25‐(OH)₂D₃ at 24 μM, 18 μM, and 12 μM and blunted by 6 μM and 3 μM. At a concentration of 12 μM, 1β,25‐(OH)₂D₃ also diminished the binding of [¹⁴C]‐1α,25‐(OH)₂D₃ bromoacetate to annexin II, but cholecalciferol, 25‐(OH)D₃, and 24,25‐(OH)₂D₃ did not. Saturation analyses of the binding of [³H]‐1α,25‐(OH)₂D₃ to purified annexin II showed a K_D of 5.5 × 10⁻⁹ M, whereas [³H]‐1β,25‐(OH)₂D₃ exhibited a K_D of 6.0 × 10⁻⁹ M. Calcium, which binds to the carboxy terminal domain of annexin II, had a concentration‐dependent effect on [¹⁴C]‐1α,25‐(OH)₂D₃ bromoacetate binding to annexin II, with 600 nM calcium being able to inhibit binding of the radiolabeled analog. The inhibitory effect of calcium was prevented by EDTA. Homocysteine, which binds to the amino terminal domain of annexin II, had no effect on the binding of the bromoacetate analog to the protein. The data indicate that 1α,25‐(OH)₂D₃ binding to annexin II is specific and suggest that the binding site may be located on the carboxy terminal domain of the protein. The ability of 1β,25‐(OH)₂D₃ to inhibit the binding of [¹⁴C]‐1α,25(OH)₂D₃ bromoacetate to annexin II provides a biochemical explanation for the ability of the 1β‐epimer to inhibit the rapid actions of the hormone in vitro. J. Cell. Biochem. 80:259–265, 2000. © 2000 Wiley‐Liss, Inc.     

A rapid and sensitive in vitro assay of 25-hydroxyvitamin D3-1 alpha-hydroxylase and 24-hydroxylase using rat kidney homogenates

A sensitive and rapid in vitro assay of 25-hydroxyvitamin D3 [25-(OH)D3]-1 alpha- and 24-hydroxylase activities was developed using rat kidney homogenates. A potent inhibitor of the enzymes in rat plasma was removed by thoroughly perfusing rats with saline. Kidney homogenates prepared from vitamin D-deficient rats preferentially produced tritiated 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] from 25(OH) [3H]D3. Addition of 10 microliter or more of rat plasma to 3 ml of 10% kidney homogenates suppressed 1 alpha-hydroxylase activity dose-dependently. Thyroparathyroidectomy (TPTX) of vitamin D-deficient rats greatly abolished 1 alpha-hydroxylase activity. Administration of parathyroid hormone to the TPTX rats increased 1 alpha-hydroxylase activity and that of 1 alpha,25(OH)2D3 enhanced 24-hydroxylase markedly. Since this assay is technically simple, rapid and sensitive, it will be useful in studying the regulatory mechanism in the renal metabolism of 25(OH)D3 in mammals.     

In vitro metabolism of 19-nor-1alpha, 25-(OH)2D2 in cultured cell lines: inducible synthesis of lipid- and water-soluble metabolites

The active vitamin D analog, 19-nor-1alpha,25-dihydroxyvitamin D2 (19-nor-1alpha,25-(OH)2D2), has a similar structure to the natural vitamin D hormone, 1a,25-dihydroxyvitamin D3 (1alpha,25-(OH)2D3), but lacks the C10-19 methylene group and possesses an ergosterol/ vitamin D2 rather than a cholesterol/vitamin D3 side chain. We have used this analog to investigate whether any of these structural features has any effect upon the type and rate of in vitro metabolism observed. Using a vitamin D-target cell, the human keratinocyte, HPK1A-ras, we observed formation of a number of metabolites, three of which were purified by extensive HPLC and conclusively identified by a combination of GC-MS and chemical derivatization as 19-nor-1alpha,24,25-(OH) 3D2, 19-nor-1alpha,24,25,26-(OH) 4D2, and 19-nor-1alpha,24,25,28-(OH)4,D2. The first metabolite is probably a product of the vitamin D-inducible cytochrome P450, P450cc24 (CYP24), while the latter two metabolites are likely to be further metabolic products of 19-nor-1alpha,24,25-(OH)3D2. These hydroxylated metabolites resemble those identified by other workers as products of the metabolism of 1alpha,25-(OH)2D2 in the perfused rat kidney. It therefore appears from the similar metabolic fate of 19-nor-1alpha,25-(OH)2D2 and 1alpha,25-(OH)2D2 that the lack of the C10-19 methylene group has little effect upon the nature of the lipid-soluble metabolic products and the rate of formation of these products seems to be comparable to that of products of 1alpha,25-(OH)2D3 in vitamin D-target cells. We also found extensive metabolism of 19-nor-1alpha,25(OH)2D2 to water-soluble metabolites in HPK1A-ras, metabolites which remain unidentified at this time. When we incubated 19-nor-1alpha,25-(OH)2D2 with the liver cell line HepG2, we obtained only 19-nor-1alpha,24,25-(OH)3D2. We conclude that 19-nor-1alpha,25-(OH)2D2 is efficiently metabolized by both vitamin D-target cells and liver cells.     

26-Hydroxylation of 1alpha,25-dihydroxyvitamin D3 does not occur under physiological conditions

The 26-hydroxylation of 1alpha,25-dihydroxyvitamin D3 in rats in vitro and in vivo was studied under physiological conditions. Incubation of 1alpha,25-dihydroxy-[26,27-3H]vitamin D3 with rat kidney or rat liver homogenate showed formation of a metabolite that was identified as 1alpha,25(S),26-trihydroxy-[26,27-3H]vitamin D3 by comigration on three different HPLC systems and a periodate cleavage reaction. This metabolite was not generated by hydroxylation of 1alpha,25-dihydroxy-[26,27-3H]vitamin D3 itself but by an enzymatic conversion of a precursor that was formed nonenzymatically in substantial amounts upon storage of 1alpha,25-dihydroxy-[26,27-3H]vitamin D3 in ethanol at -20 degrees C under argon for more than three weeks. An in vivo metabolism study in rats dosed with a physiological dose of 1alpha,25-dihydroxy-[26,27-3H]vitamin D3 confirmed the absence of 26-hydroxylation of the hormone. As expected at 6 h postinjection of purified 1alpha,25-dihydroxy-[26,27-3H]vitamin D3, 1alpha,24(R),25-trihydroxy-[26,27-3H]vitamin D3, as well as traces of (23S,25R)-1alpha,25-dihydroxy-[3H]vitamin D3-lactone were detected and identified on straight phase and reverse phase HPLC in serum, kidney, and liver.     

C-25 hydroxylation of 1alpha,24(R)-dihydroxyvitamin D3 is catalyzed by 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1): metabolism studies with human keratinocytes and rat recombinant CYP24A1

Recently, 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1) has been shown to catalyze not only hydroxylation at C-24 but also hydroxylations at C-23 and C-26 of the secosteroid hormone 1alpha, 25-dihydroxyvitamin D3 (1alpha,25(OH)2D3). It remains to be determined whether CYP24A1 has the ability to hydroxylate vitamin D3 compounds at C-25. 1alpha,24(R)-dihydroxyvitamin D3 (1alpha,24(R)(OH)2D3) is a non-25-hydroxylated synthetic vitamin D3 analog that is presently being used as an antipsoriatic drug. In the present study, we investigated the metabolism of 1alpha,24(R)(OH)2D3 in human keratinocytes in order to examine the ability of CYP24A1 to hydroxylate 1alpha,24(R)(OH)2D3 at C-25. The results indicated that keratinocytes metabolize 1alpha,24(R)(OH)2D3 into several previously known both 25-hydroxylated and non-25-hydroxylated metabolites along with two new metabolites, namely 1alpha,23,24(OH)3D3 and 1alpha,24(OH)2-23-oxo-D3. Production of the metabolites including the 25-hydroxylated ones was detectable only when CYP24A1 activity was induced in keratinocytes 1alpha,25(OH)2D3. This finding provided indirect evidence to indicate that CYP24A1 catalyzes C-25 hydroxylation of 1alpha,24(R)(OH)2D3. The final proof for this finding was obtained through our metabolism studies using highly purified recombinant rat CYP24A1 in a reconstituted system. Incubation of this system with 1alpha,24(R)(OH)2D3 resulted in the production of both 25-hydroxylated and non-25-hydroxylated metabolites. Thus, in our present study, we identified CYP24A1 as the main enzyme responsible for the metabolism of 1alpha,24(R)(OH)2D3 in human keratinocytes, and provided unequivocal evidence to indicate that the multicatalytic enzyme CYP24A1 has the ability to hydroxylate 1alpha,24(R)(OH)2D3 at C-25.     

Studies on the mechanism of action of 1 alpha, 24-dihydroxyvitamin D3. II. Specific binding of alpha, 24-dihydroxyvitamin D3 to chick intestinal receptor

The binding of vitamin D3 analogues to the chick intestinal cytosol receptor was studied. In intestinal cytosol fraction, receptor proteins having the sedimentation constant of 2.5 S and 3.7 S to which 1 alpha,25-dihydroxyvitamin D3 binds were present, and the latter was specific for the compound. The binding of 1 alpha,24(R)-dihydroxyvitamin D3 and 1 alpha,24(S)-dihydroxyvitamin D3 to the receptor was also observed, while very weak binding was seen in the case of 24(R)25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3. The binding affinity of 1 alpha,24(R)-dihydroxyvitamin D3 to the 3.7 S receptor was 1.3 times as high as that of 1 alpha,25-dihydroxyvitamin D3, whereas those of 1 alpha,24(S)-dihydroxyvitamin D3, 1 alpha-hydroxyvitamin D3 and 25-hydroxyvitamin D3 were 10, 304 and 652 times lower than 1 alpha,25-dihydroxyvitamin D3, respectively. The dissociation constant of the receptor-1 alpha,25-dihydroxyvitamin D3 complex at 0 degrees C was 3.0 x 10(-11) M, and the dissociation constants were calculated to be 2.4 x 10(-11) M and 2.7 x 10(-10) M for the complexes with 1 alpha,24(R)-dihydroxyvitamin D3 and 1 alpha,24(S)-dihydroxyvitamin D3, respectively.     

Potent inhibition of dendritic cell differentiation and maturation by vitamin D analogs

We show that the immunosuppressive effects of 1alpha, 25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) are due, in part, to inhibition of the T cell stimulatory functions of dendritic cells (DCs). Addition of 10(-12) and 10(-8) M 1alpha,25(OH)(2)D(3) to murine DC cultures resulted in a concentration-dependent reduction in levels of class II MHC and the co-stimulatory ligands B7-1, B7-2, and CD40 without affecting the number of DCs generated. Higher concentrations of 1alpha,25(OH)(2)D(3) reduced DC yield. The capacity of DCs to induce proliferation of purified allogeneic T cells was reduced by 1alpha,25(OH)(2)D(3). The vitamin D(3) analog, 1alpha,25(OH)(2)-16-ene-23-yne-26,27-hexafluoro-19-nor -D(3), exerted identical effects at 100-fold lower concentrations. Inhibition of DC maturation and stimulatory function was absent in cultures from mice genetically lacking vitamin D receptors (VDR). Vitamin D analogs effectively reduce DC function via VDR-dependent pathways.     

Effects of the vitamin D3 analog 1α,25-dihydroxyvitamin D3-3β-bromoacetate on rat osteosarcoma cells: Comparison with 1α,25-dihydroxyvitamin D3

The actions of the hormonal form of vitamin D, 1α,25-dihydroxyvitamin D₃ [1α,25-(OH)₂D₃], are mediated by both genomic and nongenomic mechanisms. Several vitamin D synthetic analogs have been developed in order to identify and characterize the site(s) of action of 1α,25-(OH)₂D₃ in many cell types including osteoblastic cells. We have compared the effects of 1α,25-(OH)₂D₃ and a novel 1α,25-(OH)₂D₃ bromoester analog (1,25-(OH)₂-BE) that covalently binds to vitamin D receptors. Rat osteosarcoma cells that possess (ROS 17/2.8) or lack (ROS 24/1) the classic intracellular vitamin D receptor were studied to investigate genomic and nongenomic actions. In ROS 17/2.8 cells plated at low density, the two vitamin D compounds (1 × 10⁻⁸ M) caused increased cell proliferation, as assessed by DNA synthesis and total cell counts. Northern blot analysis revealed that the mitogenic effect of both agents was accompanied by an increase in steady-state osteocalcin mRNA levels, but neither agent altered alkaline phosphatase mRNA levels in ROS 17/2.8 cells. ROS 17/2.8 cells responded to 1,25-(OH)₂-BE but not the natural ligand with a significant increase in osteocalcin secretion after 72, 96, 120, and 144 hr of treatment. Treatment of ROS 17/2.8 cells with the bromoester analog also resulted in a significant decrease in alkaline phosphatase-specific activity. To compare the nongenomic effects of 1α,25-(OH)₂D₃ and 1,25-(OH)₂-BE, intracellular calcium was measured in ROS 24/1 cells loaded with the fluorescent calcium indicator Quin 2. At 2 × 10⁻⁸ M, both 1α,25-(OH)₂D₃ and 1,25-(OH)₂-BE increased intracellular calcium within 5 min. Both the genomic and nongenomic actions of 1,25-(OH)₂-BE are similar to those of 1α,25-(OH)₂D₃, and since 1,25-(OH)₂-BE has more potent effects on osteoblast function than the naturally occurring ligand due to more stable binding, this novel vitamin D analog may be useful in elucidating the structure and function of cellular vitamin D receptors. © 1996 Wiley-Liss, Inc.     

Evidence for distinct membrane receptors for 1 alpha,25-(OH)(2)D(3) and 24R,25-(OH)(2)D(3) in osteoblasts

1 alpha,25-(OH)(2)D(3) exerts its effects on chondrocytes and enterocytes via nuclear receptors (1,25-nVDR) and a separate membrane receptor (1,25-mVDR) that activates protein kinase C (PKC). 24R,25-(OH)(2)D(3) also stimulates PKC in chondrocytes, but through other membrane mechanisms. This study examined the hypothesis that osteoblasts possess distinct membrane receptors for 1 alpha,25-(OH)(2)D(3) and 24R,25-(OH)(2)D(3) that are involved in the activation of PKC and that receptor expression varies as a function of cell maturation state. 1 alpha,25-(OH)(2)D(3) stimulated PKC in well differentiated (UMR-106, MC-3T3-E1) and moderately differentiated (ROS 17/2.8) osteoblast-like cells, and in cultures of fetal rat calvarial (FRC) cells and 2T3 cells treated with rhBMP-2 to promote differentiation. 24R,25-(OH)(2)D(3) stimulated PKC in FRC and 2T3 cultures that had not been treated to induce differentiation, and in ROS 17/2.8 cells. MG63 cells, a relatively undifferentiated osteoblast-like cell line, had no response to either metabolite. Ab99, a polyclonal antibody generated to the chick enterocyte 1,25-mVDR, but not a specific antibody to the 1,25-nVDR, inhibited response to 1 alpha,25-(OH)(2)D(3). 1 alpha,25-(OH)(2)D(3) exhibited specific binding to plasma membrane preparations from cells demonstrating a PKC response to this metabolite that is typical of positive cooperativity. Western blots of these membrane proteins reacted with Ab99, and the Ab99-positive protein had an Mr of 64 kDa. There was no cross-reaction with antibodies to the C- or N-terminus of annexin II. The effect of 24,25-(OH)(2)D(3) on PKC was stereospecific; 24S,25-(OH)(2)D(3) had no effect. These results demonstrate that response to 1 alpha,25-(OH)(2)D(3) and 24R,25-(OH)(2)D(3) depends on osteoblast maturation state and suggest that specific and distinct membrane receptors are involved.     

C-6 functionalized analogs of 25-hydroxyvitamin D3 and 1alpha,25-dihydroxyvitamin D3: synthesis and binding analysis with vitamin D-binding protein and vitamin D receptor.

In this article, we describe the development of a general synthetic strategy to functionalize the C-6 position of vitamin D3 and its biologically important metabolites, i.e. 25-hydroxyvitamin D3 (25-OH-D3) and 1alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3]. We employed Mazur's cyclovitamin D method to synthesize vitamin D3 analogs with several functionalities at the C-6 position. In addition, we synthesized 6-(3-hydroxypropyl) and 6-[(2-bromoacetoxy)propyl] derivatives of 25-OH-D3 15 and 16, respectively, and 6-(3-hydroxypropyl) derivative of 1,25(OH)2D3 17. Competitive binding assays of 15-17 with human serum vitamin D-binding protein showed that all these analogs specifically bound to this protein, although with significantly lower affinity than the 25-OH-D3, the strongest natural binder, but with comparable affinity with 1,25(OH)2D3, the hormone. On the other hand, 6-[3-hydroxypropyl], 1alpha,25-dihydroxyvitamin D3 17 did not show any specific binding for recombinant nuclear vitamin D receptor. These results indicated that the region containing the C-6 position of the parent seco-steroid [1,25(OH)2D3] may be an important recognition marker towards vitamin D receptor binding. Information, delineated in this article, will be important for evaluating structure-activity relationship in synthetic analogs of vitamin D and its metabolites.     

Isolation and identification of 1 alpha,25-dihydroxy-24-oxovitamin D3, 1 alpha,25-dihydroxyvitamin D3 26,23-lactone, and 1 alpha,24(S),25-trihydroxyvitamin D3: in vivo metabolites of 1 alpha,25-dihydroxyvitamin D3

Three new in vivo metabolites of 1 alpha,25-dihydroxyvitamin D3 were isolated from the serum of dogs given large doses (two doses of 1.5 mg/dog) of 1 alpha,25-dihydroxyvitamin D3. The metabolites were isolated and purified by methanol-chloroform extraction and a series of chromatographic procedures. By cochromatography on a high-performance liquid chromatograph, ultraviolet absorption spectrophotometry, mass spectrometry, Fourier-transform infrared spectrophotometry, and specific chemical reactions, the metabolites were identified as 1 alpha,25-dihydroxy-24- oxovitamin D3, 1 alpha,25-dihydroxyvitamin D3 26,23-lactone, and 1 alpha,24(S),25-trihydroxyvitamin D3. According to these procedures, the total amounts of the isolated metabolites were as follows: 1 alpha,25-dihydroxyvitamin D3, 23.6 micrograms; 1 alpha,25-dihydroxy-24- oxovitamin D3, 1.8 micrograms; 1 alpha,25-dihydroxyvitamin D3 26,23-lactone, 9.2 micrograms; 1 alpha,24(R),25-trihydroxyvitamin D3, 15.4 micrograms; 1 alpha,24(S),25-trihydroxyvitamin D3, 1.0 microgram. With recovery corrections, the serum levels of each metabolite were approximately 49 ng/mL for 1 alpha,25-dihydroxyvitamin D3, 3.7 ng/mL for 1 alpha,25-dihydroxy-24- oxovitamin D3, 19 ng/mL for 1 alpha,25-dihydroxyvitamin D3 26,23-lactone, 32 ng/mL for 1 alpha,24(R),25-trihydroxyvitamin D3, and 2.1 ng/mL for 1 alpha,24(S),25-trihydroxyvitamin D3.     

1 alpha, 25-Dihydroxyvitamin D3 and analogues of vitamin D3 induce alkaline phosphatase activity in osteoblastic cells derived from newborn mouse calvaria

In order to study the effects of vitamin D metabolites on bone metabolism, clone MC3T3-E1 cells, which have retained osteoblastic activity, were cultured with various concentrations of the hormone, 1 alpha, 25-dihydroxyvitamin D3 [1 alpha, 25 (OH)2D3]. A physiological concentration of 1 alpha, 25 (OH)2D3 stimulated alkaline phosphatase (ALP) activity in the cells. Other metabolites--1 alpha, 24-dihydroxyvitamin D3 [1 alpha, 24 (OH)2D3], 1 alpha-hydroxyvitamin D3 [1 alpha (OH)D3], and 24R,25-dihydroxyvitamin D3 [24R,25 (OH)2D3]--also induced increases in ALP activity in a dose-dependent fashion. However, their effective concentrations were 100 or 1,000 times greater than that of 1 alpha, 25 (OH)2D3. Hormone-induced and native ALP activities in the cells were of the same type as that found in newborn mouse calvaria; that is, they were heat-labile, L-homoarginine- and levamisole-sensitive, and L-phenylalanine-insensitive (liver-bone-kidney type). These results show that vitamin D metabolites stimulate bone formation in vitro and that they may be involved in bone formation in vivo as well.     

Isolation and characterization of 1 alpha-hydroxy-23-carboxytetranorvitamin D: a major metabolite of 1,25-dihydroxyvitamin D3.

The in vivo side-chain oxidation of 1 alpha,25-dihydroxyvitamin D3 was investigated by using a double-label radiotracer technique. Rats dosed with 1 alpha,25-dihydroxy-[3 alpha-3H]vitamin D3 and 1 alpha,25-dihydroxy[26,27-14C]vitamin D3 produced compounds with a high 3H/14C ratio. These compounds were found in sizable quantities in intestine and liver within 3 h after dosing. The major side-chain oxidized metabolite migrated as an acid on DEAE-Sephadex chromatography and contained no 14C. Methyl esterification of this compound with diazomethane proceeded in good yield and rendered the compound more amenable to chromatographic purification. The metabolite was isolated in several steps from rats dosed with 1 microgram of 1 alpha,25-dihydroxy[3 alpha-3H]vitamin D3. The metabolite was obtained in pure form as the methyl ester and was positively identified as 1 alpha,3 beta-dihydroxy-24-nor-9,10-seco-5,7,10(19)cholatrien-23-oic acid. The trivial name calcitroic acid is proposed for this major side-chain oxidized metabolite of 1,25-dihydroxyvitamin D3.     

1 alpha, 25-dihydroxyvitamin D3 specific regulation of growth, morphology, and fibronectin in a human osteosarcoma cell line

The ability of the hormonally active vitamin D metabolite, 1 alpha, 25-dihydroxyvitamin D3, to affect cell growth, morphology and fibronectin production has been examined using the MG-63 human osteosarcoma cell line. Hormone treatment reduced cell growth rate, saturation density and [3H]thymidine incorporation. Inhibition was specific for 1 alpha, 25-dihydroxyvitamin D3 relative to other vitamin D metabolites (1 alpha, 25-dihydroxyvitamin D3 greater than 25-dihydroxyvitamin D3 greater than 24R,25-dihydroxyvitamin D3 greater than D3), antagonized by high concentrations of serum and readily reversed by removal of 1 alpha, 25-dihydroxyvitamin D3 from the culture medium. Hormone treatment also increased cell associated alkaline phosphatase activity up to twofold and altered morphology such that treated cells were more spread out on the culture dish and contained more cytoplasmic processes. Significantly, 1 alpha, 25-dihydroxyvitamin D3 increased cellular and medium concentrations of fibronectin, a glycoprotein known to be involved in cellular adhesiveness. MG-63 cells contain a specific 1 alpha, 25-dihydroxyvitamin D3 receptor which may mediate these responses.